ACTA BIOLOGICA CRACOVIENSIA Series Botanica 54/1: 105–112, 2012 DOI: 10.2478/v10182-012-0012-0

FLAVONOID AND ORGANIC ACID CONTENT IN HIPS (ROSA L., SECT. CANINAE DC. EM. CHRIST.)

ARTUR ADAMCZAK1*, WALDEMAR BUCHWALD1, JERZY ZIELIŃSKI2, AND SEBASTIAN MIELCAREK1

1Institute of Natural Fibres and Medicinal , Wojska Polskiego 71B, 60-630 Poznań, Poland 2Department of Forestry Natural Foundation, Poznań University of Life Sciences, Wojska Polskiego 71D, 60-625 Poznań, Poland

Received June 16, 2011; revision accepted June 29, 2012

We determined the level of flavonoids, citric acid and ascorbic acid in hips of rose species from the Caninae sec- tion occurring in Poland. We performed phytochemical analyses of 75 samples representing 11 species: Savi, R. canina L., R. dumalis Bechst., R. glauca Pourret, R. inodora Fries, R. jundzillii Besser, R. rubiginosa L., R. sherardii Davies, R. tomentosa Sm., R. villosa L. and R. zalana Wiesb. Flavonoid content was determined spectrophotometrically, and organic acid concentrations by HPLC. The content of the studied compounds varied greatly. Interspecific differences in the amount of flavonoids and ascorbic acid were highly significant. The most common species, , showed low average content of vitamin C (0.51 g/100 g of dry matter) and flavonoids (41 mg/100 g DM) and high content of citric acid (3.48 g/100 g DM). Ascorbic acid was highest in R. villosa hips (avg. 2.25 g/100 g DM), flavonoids were highest in R. rubiginosa (72 mg/100 g DM), and citric acid was highest in R. tomentosa (4.34 g/100 g DM). Flavonoid level correlated negatively with the amount of citric acid (r=-0.47, p<0.001). Cluster analysis of rose species based on the content of the investi- gated compounds confirmed the validity of the division of sect. Caninae into three subsections: Rubiginosae, Vestitae and Rubrifoliae. The phytochemical variation of these reflects their probable phylogenetic rela- tionships as determined from morphology. Key words: Rosa, Caninae, rose hips, medicinal plants, flavonoids, vitamin C, phylogenetic rela- tionships, .

INTRODUCTION polyploids of hybrid origin, distinguishable by a spe- cific process of (so-called balanced het- extracts have antibacterial, antifungal and erogamy). Presumably this section is at a relatively antiinflammatory properties (Trovato et al., 2000; early stage of divergence, since the differences Kumarasamy et al., 2002; Larsen et al., 2003), and between species are sometimes smaller than their antioxidative activity has also been shown (Gao et intraspecific variability. Frequently it is only possi- al., 2000; Moure et al., 2001; Ochmańska et al., ble to separate particular taxa on the basis of a 2001; van Rensburg, 2005). They contain large whole set of morphological characters. Other prob- amounts of pharmacologically active compounds lems with identification of species are related to the such as organic acids (including ascorbic acid – vita- fact that they undergo secondary hybridization, pro- min C), flavonoids, and tannins (Gao et ducing numerous transitional forms (Zieliński, al., 2005; Novruzov and Shamsizade, 2005; Olsson 1985, 1987; Popek, 1996; Henker, 2000; Werle- et al., 2005; Nowak, 2006; Buchwald et al., 2007; mark and Nybom, 2001; Ritz and Wissemann, Kozłowski et al., 2009). 2003; Lim et al., 2005; Ritz et al., 2005; Wissemann A large majority of European rose species et al., 2007). Phytochemical data, which may serve belong to the section Caninae DC. em. Christ. This as an additional taxonomic criterion, are an impor- is the most polymorphic section of the genus, posing tant supplemental tool for differentiation within this great difficulties in their taxonomy. It comprises group (Stace, 1993).

*e-mail: [email protected]

PL ISSN 0001-5296 © Polish Academy of Sciences and Jagiellonian University, Cracow 2012 106 Adamczak et al.

The published phytochemical studies of roses R. zalana Wiesb. Samples were collected in include analyses of the chemical composition of 2007–2008 in Greater Poland (Wielkopolska), petals (Biolley and Jay, 1993; Jay et al., 1994; Lower Silesia (Dolny Śląsk), the Lubusz region Raymond et al., 1995), hypanthia (Ercisli, 2007; (Ziemia Lubuska), the Ponidzie region (Ponidzie) Nojavan et al., 2008; Adamczak et al., 2010), ach- and the Kraków-Częstochowa Upland (Wyżyna enes (Özcan, 2002; Kumarasamy et al., 2003; Krakowsko-Częstochowska). Rose hips were har- Nowak, 2005) and (Krzaczek and Krzaczek, vested from the end of August to mid-October 1979; Tarnoveanu et al., 1995; Nowak and Gawlik- depending on the ripening period (Zieliński, 1987; Dziki, 2007). In the hips of roses from the Caninae Kovács et al., 2005). They were picked at full matu- section, the level of ascorbic acid has been deter- rity as judged by their color (Erentürk et al., 2005; mined most frequently (Halásová and Jièínská, Gao et al., 2005; Nojavan et al., 2008). The material 1988; Gao et al., 2000; Kovács et al., 2000; Demir was taken mainly from wild specimens, and in sev- and Özcan, 2001; Strålsjö et al., 2003; Uggla et al., eral cases from in the garden collection of 2003; Erentürk et al., 2005; Kovács et al., 2005; the Institute of Dendrology, Polish Academy of Novruzov and Shamsizade, 2005; Pirone et al., Sciences in Kórnik near Poznań. The obtained rose 2007; Nojavan et al., 2008; Nueleanu et al., 2008). hips were lyophilized at -50°C and 0.5 hPa (Heto Also measured were carotenoids (Hodisan et al., Drywinner DW 30). 1997; Gao et al., 2000; Böhm et al., 2003; Novruzov Species identification and taxon nomenclature and Shamsizade, 2005; Olsson et al., 2005; Pirone follow Zieliński (1987). All samples are documented et al., 2007), flavonoids and anthocyanins (Nowak, by voucher specimens deposited in the herbarium of 1994; Nowak and Hawrył, 2005; Pirone et al., 2007; the Department of Botany, Breeding and Agronomy, Adamczak et al., 2010), total phenols (Gao et al., Institute of Natural Fibres and Medicinal Plants in 2000; Nowak and Gawlik-Dziki, 2004; Olsson et al., Poznań. 2005; Ercisli, 2007; Kilicgun and Altiner, 2010), sugars (Kovács et al., 2000; Uggla et al., 2005; FLAVONOID ANALYSIS Pirone et al., 2007) and nutrients (Kovács et al., 2000; Demir and Özcan, 2001; Ercisli, 2007). The Phytochemical screening of total flavonoids in largest amount of such data is in a monographic lyophilized plant material (2.00 g sample) was done study by Nowak (2006). However, these studies using the methodology described in the 6th most often relate only to Rosa canina (Hodisan et Farmakopea Polska (2002). The spectrophotomet- al., 1997; Demir and Özcan, 2001; Hvattum, 2002; ric method we used to quantify total flavonoids, Erentürk et al., 2005; Novruzov and Shamsizade, expressed as quercetin equivalent, is the standard 2005; Nojavan et al., 2008; Nueleanu et al., 2008; Christ-Müller's procedure described for medicinal Kilicgun and Altiner, 2010). There are few compara- plants (Vladimir-Knežević et al., 2011). This method tive studies shedding light on intra- and interspecif- involves acid hydrolysis of flavonol glycosides and ic phytochemical variability within sect. Caninae then the formation of colored complexes of these (Krzaczek et al., 1970; Halásová and Jièínská, 1988; flavonoid compounds with AlCl3. Absorbance was Nowak, 2006). measured at λ=425.0 nm with a Cintra 20 UV-VIS Here we determined the levels of flavonoids and spectrometer (GBC). All solvents (analytical grade) organic (citric and ascorbic) acids in hips of rose were purchased from POCH S.A., Poland. species from the Caninae section found in Poland. We assessed the taxonomic value of these groups of CITRIC AND L-ASCORBIC ACID ANALYSIS compounds based on a large number of samples and species. The freeze-dried and powdered rose hips (0.40–0.50 g) were extracted twice for 30 min with 2.5 ml 4.0% (m/V) L-cysteine and 10.0 ml water by sonification. MATERIALS AND METHODS All aqueous extracts were combined and diluted with water to 25 ml. The samples were analyzed using HPLC (PN-EN 14130:2004). PLANT MATERIAL HPLC analyses were done with an Agilent 1100 Phytochemical analysis was performed on 75 sam- HPLC system with a photodiode array detector ples of rose hips representing 11 species from sect. (DAD), and all separations were on a Lichrospher Caninae DC. em. Christ.: Rosa agrestis Savi, 100 RP18 column (250.0×4.0 mm, 5.0 μm; Merck). R. canina L., R. dumalis Bechst. (= R. glauca Vill., The mobile phase consisted of 0.0272 g/l KH2PO4 R. vosagiaca Desp.), R. glauca Pourret, R. inodora adjusted to pH 2.40 with H3PO4, applied in isocrat- Fries (= R. elliptica Tausch), R. jundzillii Besser ic elution for 30 min. The flow rate was adjusted to (= R. trachyphylla Rau.), R. rubiginosa L., R. sher- 1.0 ml/min. The detection wavelength was set to ardii Davies, R. tomentosa Sm., R. villosa L. and DAD at λ=215.0 nm for citric acid and λ=254.0 nm Flavonoids and organic acids in rose hips 107

TABLE 1. Content of flavonoids, citric acid and ascorbic acid in freeze-dried hips of roses from sect. Caninae

DM – dry matter of freeze-dried hips; content of flavonoids as quercetin equivalent; SD – standard deviation; V – variability coefficient; N – number of samples. for ascorbic acid (L-ascorbic acid). 20.0 μl samples RESULTS were injected. All separations were performed at 24.0°C. Peaks were assigned by spiking the samples Our results show large variability of the content of with standard compounds and comparing the UV the studied compounds in the hips of roses from spectra and retention times (ascorbic acid 5.66 min, sect. Caninae (Tab. 1). The observed interspecific citric acid 8.90 min). Calibration curves were differences in the amount of flavonoids (Fig. 1) and obtained from 5 concentrations of each external ascorbic acid (Fig. 2) were highly significant. For standard (0.01–1.20 mg/ml). The regression coeffi- example, in the most common species, for which the cient (R2) of the calibration curve for ascorbic acid largest number of samples was collected, average (Y = 83780x – 19.693) was 0.9985, and for citric flavonoid content in R. canina hips (41 mg/100 g acid (Y = 2003.6x + 12.795) it was R2 = 0.9998. DM) was nearly half that of R. rubiginosa hips The RSD values for the repeatability (n=4) of stan- (72 mg/100 g DM). The average amount of vitamin C dard solution were 0.40% (0.01 mg/ml ascorbic in R. canina hips (0.51 g/100 g DM) was nearly a acid) and 0.83% (0.41 mg/ml citric acid). The limits third that of R. dumalis hips (1.44 g/100 g DM). For of quantitation (LOQ) and detection (LOD) of ascor- citric acid there were no such large interspecific dif- bic acid were 0.18 and 0.06 mg/L, respectively, and ferences between the taxa with the highest number 7.41 and 2.47 mg/L for citric acid. All solvents used of samples, hence there were no statistically signifi- were HPLC grade (Merck). Reference standards cant differences (Fig. 3). The level of flavonoids in were obtained from Sigma-Aldrich. rose hips was negatively correlated with citric acid

STATISTICAL ANALYSIS Statistica 7.1 (StatSoft, 2005) was used for statisti- cal calculations. The Kruskal-Wallis test and the median test as well as post-hoc multiple compar- isons of mean ranks for all groups were used to determine the statistical significance of interspecific differences in the content of the investigated com- pounds. Pearson’s coefficient of correlation was used to evaluate correlations between variables. The Shapiro-Wilk test was applied to assess the normal- ity of variable distribution. Root transformation was performed for the right-skewed distribution of flavonoids. The phytochemical similarity of rose species was determined based on cluster analysis of standardized mean content of the studied com- pounds in the hips of particular taxa. Euclidean dis- tance was used as a measure of distance, and UPGMA as the clustering method. Fig. 1. The level of the compounds was determined per Flavonoid content (as quercetin equivalent) in freeze-dried hips of rose species from sect. Caninae the total weight of rose hips (hypanthia and ach- (n=74). Kruskal-Wallis test: 27.54, p=0.0021; median enes). Flavonoid content is given as mg/100 g, and test: 28.02, p=0.0018; values with different letters differ ascorbic and citric acid content as g/100 g rose hip significantly at p<0.05. R_ag – Rosa agrestis (n=7); R_ca dry matter (DM). The relative amount of a given – R. canina (n=12); R_du – R. dumalis (n=16); R_gl – R. compound in the hips of particular rose species is glauca (n=2); R_in – R. inodora (n=5); R_ju – R. jundzil- given as a percentage of the highest average content lii (n=3); R_ru – R. rubiginosa (n=13); R_sh – R. sher- of it found in the investigated taxa. ardii (n=9); R_to – R. tomentosa (n=4); R_vi – R. villosa (n=2); R_za – R. zalana (n=1). 108 Adamczak et al.

Fig. 2. Content of ascorbic acid (vitamin C) in freeze-dried Fig. 3. Citric acid content in freeze-dried hips of rose hips of rose species from sect. Caninae (n=75). Kruskal- species from sect. Caninae (n=75). Kruskal-Wallis test: Wallis test: 37.76, p<0.001; median test: 30.06, p<0.001; 17.70, p=0.0603 (N.S.); median test: 13.19, p=0.2131 values with different letters differ significantly at p<0.05. (N.S.); R_ag – Rosa agrestis (n=7); R_ca – R. canina R_ag – Rosa agrestis (n=7); R_ca – R. canina (n=12); (n=12); R_du – R. dumalis (n=16); R_gl – R. glauca R_du – R. dumalis (n=16); R_gl – R. glauca (n=2); R_in – (n=2); R_in – R. inodora (n=5); R_ju – R. jundzillii (n=3); R. inodora (n=5); R_ju – R. jundzillii (n=3); R_ru – R_ru – R. rubiginosa (n=13); R_sh – R. sherardii (n=10); R. rubiginosa (n=13); R_sh – R. sherardii (n=10); R_to – R_to – R. tomentosa (n=4); R_vi – R. villosa (n=2); R_za R. tomentosa (n=4); R_vi – R. villosa (n=2); R_za – – R. zalana (n=1). R. zalana (n=1).

Fig. 4. Correlation between the citric acid and flavonoid content in freeze-dried rose hips. Pearson coefficient of corre- lation: -0.47; p<0.001; n=74. Flavonoids and organic acids in rose hips 109

Fig. 5. UPGMA cluster analysis based on Euclidean dis- Fig. 6. Relative content* of flavonoids, citric acid and tance of mean content of flavonoids, citric acid and ascor- ascorbic acid in freeze-dried hips of rose species from bic acid in freeze-dried hips of rose species from sect. sect. Caninae (n=224). *Species with the highest average Caninae (n=224). content of a given compound (group of compounds) were taken as 100%. R_ag – Rosa agrestis (n=21); R_ca – R. canina (n=36); R_du – R. dumalis (n=48); R_gl – R. glau- ca (n=6); R_in – R. inodora (n=15); R_ju – R. jundzillii content (r=-0.47, p<0.001; Fig. 4). Between the (n=9); R_ru – R. rubiginosa (n=39); R_sh – R. sherardii amounts of citric acid and ascorbic acid there was a (n=29); R_to – R. tomentosa (n=12); R_vi – R. villosa statistically significant though distinctly weaker cor- (n=6); R_za – R. zalana (n=3). relation (r=0.28, p<0.05). Based on phytochemical analysis of the rose hips we constructed a dendrogram of similarity other organic acids (e.g., citric acid, commonly used between the taxa (Fig. 5), which separated three in food processing as an acidity regulator and groups of species: Rosa agrestis–R. rubiginosa– ) and flavonoids. Among the flavonoids R. inodora; Rosa canina–R. jundzillii–R. zalana; in plants, quercetin and kaempferol are important and –R. sherardii–R. villosa. The first and most common. These active compounds usual- group was distinguished primarily by having the ly occur as glycosides with the sugar moiety bound highest relative flavonoid content (avg. 91%), with at the C-3 position. In rose hips there are mainly gly- moderate levels of citric acid (60%) and ascorbic coside derivatives of quercetin: quercitrin (quer- acid (40%) (Fig. 6). The Rosa canina group was cetin-3-O-rhamnoside), isoquercitrin (quercetin-3- characterized by moderate content of flavonoids O-glucoside) and hyperoside (quercetin-3-O-galacto- (56%) and citric acid (67%) as well as low ascorbic side) (Gao et al., 2000; Hvattum, 2002; Nowak, acid content (24%). The Rosa dumalis group 2006). There may be interspecific differences showed moderate content of flavonoids (67%) and between roses in the composition and concentration higher content of citric acid (78%) and ascorbic acid of individual flavonol compounds, but this is not yet (72%). well documented (Novruzov, 2005; Nowak and Tuzimski, 2005). The literature data show large differences in the DISCUSSION content of these compounds in the hips of roses from sect. Caninae (Kovács et al., 2005; Nowak, The medicinal value of rose hips depends largely on 2006; Adamczak et al., 2010). Rosa villosa is the the content of vitamin C and flavonoids (Kuźnicka richest source of vitamin C, while R. canina usually and Dziak, 1987). This plant material is generally shows low content of it (Krzaczek et al., 1970; Gao considered to be the most abundant natural source et al., 2000); our results support that. Average of vitamin C (Erentürk et al., 2005; Kobus et al., ascorbic acid content in R. villosa was 2.25 g/100 g 2005; Nowak, 2006). The flavonoids and organic DM, and in R. canina only 0.51 g/100 g DM. R. cani- acids found in rose hips inhibit oxidation of vitamin na showed a low range of variability as compared to C, which additionally increases its stability and other species we studied (e.g., R. inodora, R. sher- bioavailability in humans (Ochmańska et al., 2001; ardii, R. dumalis). Halásová and Jièínská (1988) Padayatty and Levine, 2001; Kobus et al., 2005). found great variation of ascorbic acid content within From the point of view of phytotherapy it is impor- R. canina L. [in the broad view of this species adopt- tant to determine the content of vitamin C but also ed here, following Zieliński (1987) and Popek 110 Adamczak et al.

(1996)], ranging from 0.62 g/100 g DM in R. ande- R. glauca). There were also certain deviations from gavensis Bast. (forms with glandular pedicels) to the classification based on morphological charac- 4.57 g/100 g DM in air-dried hips without achenes in ters. Rosa zalana, which is considered to be inter- R. corymbifera Borkh. (forms with hairy leaves). mediate between R. rubiginosa and R. agrestis Those authors stressed that the level of vitamin C in (Zieliński, 1987), was outside the group of species rose hips depends largely on soil and climatic con- from the Rubiginosae subsection. R. tomentosa was ditions, but Krzaczek et al. (1970) maintained that not included with R. sherardii and R. villosa, where- the taxonomic assignment level lower than species as R. dumalis was grouped with these species. (i.e., subspecies and variety) plays a greater role. We R. dumalis has certain features (persistent sepals, found high citric acid content in R. canina hips (Fig. a wide orifice, a flat disc, erect stems) that make it 3), as did Kovács et al. (2005). R. rubiginosa hips more similar to R. sherardii of subsect. Vestitae showed the highest average flavonoid content, as in than to R. canina (caducous sepals, narrow orifice, a preliminary study (Adamczak et al., 2010); usual- conical disc, arched stems) of subsect. Caninae. ly it was low in R. canina (Fig. 1). Morphologically, R. tomentosa is intermediate Owing to difficulties in the classification and between R. canina (Caninae) and R. sherardii identification of individual rose species, herbal raw (Vestitae). material is obtained from different taxa; this is per- The variation of flavonoid and organic acid mitted by both Farmakopea Polska (1954, 1970) content in these roses (Fig. 5) well reflects the and the European Pharmacopoeia (2002). The one probable phylogenetic relationships between the usually collected is the widespread dog rose (Rosa taxa as determined from morphology. The results canina L.). Our results show that this species is not of flavonoid analyses (Fig. 1) are closest to the pat- the most valuable herbal raw material, at least as a tern of morphological data. Phenolics, especially source of vitamin C and flavonoids. The use value of flavonoids, are particularly important in chemotax- other rose species of the Caninae section is higher: onomy (Stace, 1993; Novruzov, 2005; Nowak, R. villosa (for vitamin C) and R. rubiginosa (for 2006). Not only qualitative but also quantitative flavonoids). We determined the content of the active features have chemotaxonomic value, though they compounds in whole rose pseudofruits (hips). The are more subject to environmental effects and are main part of the hip is fruit flesh. According to more difficult to interpret. Different groups of com- Kovács et al. (2000), achenes can constitute from pounds give varying results on the level of intra- 19% (Rosa agrestis) to 31% (R. micrantha) of rose and interspecific variability within the Caninae hip weight, depending on the species. Uggla et al. section (Krzaczek et al., 1970; Olsson et al., 2005; (2003) reported a positive correlation between the Nowak, 2006). Hence the largest possible number percentage of fruit flesh and the weight of whole rose of compounds should be included in such chemo- hips. taxonomic analyses, requiring us to expand and Based on their morphological characters, the continue this study. rose species of sect. Caninae that occur in Poland can be classified in five subsections (Konèalová and Klášterský, 1978; Małecka et al., 1990; Wissemann, ACKNOWLEDGEMENTS 2000; de Cock et al., 2008): Rubrifoliae (Rosa glau- ca), Caninae (R. canina, R. dumalis), Vestitae (R. We thank Prof. Ryszard Popek (Pedagogical tomentosa, R. sherardii, R. villosa), Rubiginosae University of Cracow) and Dr. Renata Piwowarczyk (R. agrestis, R. inodora, R. micrantha, R. rubigi- and Dr. Alojzy Przemyski (Jan Kochanowski nosa, R. zalana) and Trachyphyllae (R. jundzillii). University of Humanities and Sciences, Kielce) for The boundaries between these groups are not sharp, their help in field work, and Bogna Opala and since the most common species, Rosa canina, is Agnieszka Gryszczyńska (Institute of Natural Fibres morphologically linked to both R. dumalis and and Medicinal Plants, Poznań) for their help with species representing other subsections: R. tomen- phytochemical analyses. This study was financed by tosa, R. agrestis and R. jundzillii (Zieliński, 1987). the Ministry of Science and Higher Education Nevertheless, genetic investigations provide at least (research project no. N312 023 32/1556). partial confirmation of that classification (Olsson et al., 2000; Werlemark and Nybom, 2001; Nowak and Polok, 2005; de Cock et al., 2008), and it is largely REFERENCES in agreement with our results on phytochemical variability (Figs. 5, 6). Cluster analysis on the basis ADAMCZAK A, BUCHWALD W, ZIELIŃSKI J, and MIELCAREK S. 2010. of flavonoid and organic acid content separated The effect of air and freeze drying on the content of three subsections: Rubiginosae (with R. agrestis, flavonoids, β-carotene and organic acids in European R. rubiginosa and R. inodora), Vestitae (with dog rose hips (Rosa L. sect. Caninae DC. em. Christ.). R. sherardii and R. villosa) and Rubrifoliae (with Herba Polonica 56(1): 7–18. Flavonoids and organic acids in rose hips 111

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